PROJECT SUMMARY The requested high-throughput high-resolution cryo-electron microscope (FEI Krios) will replace an earlier generation instrument (FEI Polara, equipped with a Falcon 2 direct electron detecting (DED) camera and automated data collection software). Numerous projects have benefitted enormously from access to the Polara. Density maps of small virus capsids, for example, provided novel structural insights into the processes governing capsid assembly, maturation, binding to antibodies and receptors, and the consequent changes as part of the infection process. However, the Polara is aging and has a troubled service history, and significant limitations in its 20-year-old design do not support the demands of current 24/7 automated operation. In just eight years the FEI Krios has already out-sold the Polara by more than 3-to-1, demonstrating its popularity and success. It would be fair to say that, together with DED camera technology, the Krios has revolutionized protein structure determination by cryo-EM. Capabilities beyond those of the Polara that are essential for the future of cryo-EM in Pittsburgh include: robust and reliable operation; higher rate of data collection; additional lens control to aid in high resolution image quality (coherence) and image quantity; streamlined cryo-sample insertion to reduce operator error; and a new phase-plate option that is expected to be critical for achieving atomic-resolution structures of large complexes. These factors have become increasingly critical over the past 12 years of operating the Polara and together with the deep experience gained on the Polara that will transfer directly to operation of the Krios, provide strong justification for the proposed upgrade. The capabilities of the Krios will greatly impact existing projects by improving data quality and quantity. Atomic resolution structures of massive protein complexes such as herpesvirus and other large virus capsids will not be achievable without routine access to the high coherency and phase-plate capabilities of the Krios. In addition, a near 2-fold increase in data collection rate over the Polara will add critical capacity for new projects. A benefit of our Polara experience is that the Krios operating environment will be very familiar, allowing an immediate start to exploiting the power of the new microscope. The Falcon camera will be moved onto the new instrument, and the years of experience gained using the automated data collection software suites, EPU and SerialEM, will likewise transfer to the new instrument. The improved structural resolution will enable deep understanding of how the proteins work as individual subunits and together as a functioning machine. With the knowledge of how proteins function, and which parts are exposed or buried at interfaces, disease processes can be modeled and drugs designed and targeted specifically as therapy.